Molecular mechanism underlying non-discriminatory recognition of relaxin-3 by RXFP3 and RXFP4

The human relaxin family peptide receptors RXFP3 and RXFP4 play important physiological roles through interactions with endogenous hormones, relaxin-3 and insulin-like peptide 5 (INSL5). They are implicated in certain neurological and metabolic disorders. While INSL5 only activates RXFP4, relaxin-3 is recognized by both receptors. Here, we report the cryo-electron microscopy structures of RXFP3-G_i complexes bound by relaxin-3 or a small-molecule dual agonist (compound 4), and relaxin-3 in complex with RXFP4-G_i, with global resolutions of 2.91 Å, 2.95 Å, and 3.10 Å, respectively. It is found that relaxin-3 adopts a conserved binding conformation within the transmembrane domain (TMD) bundle of RXFP3 and RXFP4, where the C-terminal tip residues of its B chain, R26 and W27, make extensive contacts with conserved receptor residues, thereby activating RXFP3 and RXFP4. Compound 4 mimics these key interactions by binding to both receptors. In contrast, the C-terminal residues composition and TMD-binding angle of INSL5 in RXFP4 differ significantly from that of relaxin-3, ensuring its selectivity for RXFP4. These findings deepen our understanding about the structural basis of ligand recognition and selectivity in this G protein-coupled receptor subfamily.